Artificial chromosomes comprising EHV sequences

ABSTRACT

The invention belongs to the field of animal health, in particular equine diseases caused by equine herpesvirus (EHV). The invention relates to artificial chromosomes comprising the genome of equine herpesviruses, methods of producing attenuated or virulent EHV with or without the insertion of foreign genes, EHV obtainable with said methods and pharmaceutical compositions comprising said viruses.

[0001] The invention belongs to the field of animal health, inparticular equine diseases caused by equine herpesvirus (EHV). Theinvention relates to artificial chromosomes comprising the genome ofequine herpesviruses, methods of producing attenuated EHV viruses, EHVviruses obtainable with said methods and pharmaceutical compositionscomprising said viruses.

BACKGROUND OF THE INVENTION

[0002] Equine herpesvirus 1 (EHV-1), a member of the Alphaherpesvirinae,is the major cause of virus-induced abortion in equids and causesrespiratory and neurological disease. The entire DNA sequence of theEHV-1 strain Ab4p has been determined (Telford, E. A. R. et al., 1992).Only few genes and gene products have been characterized for theirrelevance for the virulence or immunogenicity of EHV-1 because theproduction of viral mutants is still relying on the generation ofrecombinant viruses by homologous recombination between the viral genomeand respective foreign DNA to be inserted in cultured mammalian cells.

[0003] For control of EHV-1 infections, two different approaches arefollowed. First, modified live vaccines (MLVs) have been developed,including the strain RacH (Mayr, A. et al., 1968; Hübert, P. H. et al.,1996), which is widely used in Europe and the United States. Second,inactivated vaccines and independently expressed viral glycoproteinshave been assessed for their immunogenic and protective potential. Amongthe glycoproteins that were expressed using recombinant baculovirusesare the glycoproteins (g) B, C, D, and H, which induced partialprotection against subsequent challenge EHV-1 infection in a murinemodel (Awan, A. R. et al., 1990; Tewari, D. et al., 1994; Osterrieder,N. et al., 1995; Stokes, A. et al., 1996). However, the use of MLVs hasadvantages over killed and subunit vaccines. MLVs are highly efficientin inducing cell-mediated immune responses, which are most likely to beresponsible for protection against disease (Allen, G. P. et al., 1995;Mumford, J. A. et al., 1995).

[0004] Herpesvirus glycoproteins are crucially involved in the earlystages of infection, in the release of virions from cells, and in thedirect cell-to-cell spread of virions by fusion of neighboring cells. Todate, 11 herpes simplex virus type 1 (HSV-1)-encoded glycoproteins havebeen identified and have been designated gB, gC, gD, gE, gG, gH, gI, gJ,gK, gL, and gM. HSV-1 mutants lacking gC, gE, gG, gI, gJ, and gM areviable, indicating that these genes are dispensable for replication incultured cells. Comparison of the HSV-1 and equine herpesvirus 1nucleotide sequences revealed that all of the known HSV-1 glycoproteinsare conserved in EHV-1. According to the current nomenclature, theseglycoproteins are designated by the names of their HSV-1 homologs. Inaddition, a further envelope protein named gp1/2 and a tegument protein,the VP13/14 homolog of HSV-1, have been described to be glycosylated incase of EHV-1 (reviewed in Osterrieder et al., 1998). It is known thatEHV-1 gC, gE gI, and gM are not essential for growth in cell culture,whereas gB and gD appear to be essential for virus growth in culturedcells. The contributions of other EHV-1 glycoproteins to replication incultured cells are not known (Neubauer et al., 1997; Flowers et al.,1992).

[0005] The gp1/2 glycoprotein is encoded by gene 71 (Wellington et al.,1996; Telford et al., 1992) and was also shown to be nonessential forvirus growth in vitro (Sun et al., 1996). In addition, a viral mutantcarrying a lacZ insertion in the gene 71 open reading frame wasapathogenic in a murine model of infection but still able to preventagainst subsequent challenge infection (Sun et al., 1996; Marahall etal. 1997). In addition, the KyA strain of EHV-1 harbors a major deletionin the coding sequences of gene 71 (Colle et al., 1996).

[0006] The technical problem underlying this invention was to provide anew tool and procedure to generate attenuated equine herpesviruses ofdefined specificity.

SUMMARY OF THE INVENTION

[0007] The above-captioned technical problem is solved by theembodiments characterized in the claims and the description.

[0008] The invention relates to artificial chromosomes comprising thegenome of EHV, methods of producing attenuated EHV, EHV obtainable withsaid methods and pharmaceutical compositions comprising said viruses.

FIGURE LEGENDS

[0009]FIG. 1:

[0010] Cloning strategy for introduction of mini F plasmid sequencesinto the RacH genome (A). PCR amplification of fragments bordering gene71 located in the US region of the genome (B) was done and the resultingBamHI-KpnI and SalI-SphI fragments were consecutively cloned into vectorpTZ18R (C). Mini F plasmid sequences were released from recombinantplasmid pHA2 (Adler et al., 2000) with PacI and cloned to give rise torecombinant plasmid p71-pHA2 (D). This plasmid was co-transfected withRacH DNA into RK13 cells and fluorescing virus progeny was selected.Viral DNA from green fluorescing virus progeny was used to transformEscherichia coli DH10B cells from which infectious RacH-BAC wasisolated. Restriction enzyme sites and scales (in kbp) are given.

[0011]FIG. 2:

[0012] Restriction enzyme digests of RacH and RacH-BAC. After separationby 0.8% agarose gel electrophoresis, fragments were transferred to anylon membrane (Pharmacia-Amersham) and hybridized with a labelled pHA2probe (see FIG. 1). Reactive fragments which are present due toinsertion of mini F plasmid sequences are indicated by asterisks.Molecular weight marker is the 1 kb ladder (Gibco-BRL). The restrictionenzymes used are indicated.

[0013]FIG. 3:

[0014] Plaque sizes of RacH and RacH-BAC. Plaque sizes were determinedon RK13 cells by measuring diameters of 150 plaques each. Plaque sizesof RacH were set to 100%, respectively, and plaque sizes of virusprogeny reconstituted from BAC were compared to those of the parentalvirus. Standard deviations are given.

[0015]FIG. 4:

[0016] Principle of the deletion of the genes encoding for gD (a) or gM(b) in RacH-BAC by replacing the open reading frames with the kanamycinresistance gene (kan^(R)) using E/T cloning. The kan^(R) gene wasamplified by PCR using the primers listed in Table 1, and the ampliconwas electroporated into DH10B cells containing RacH-BAC and plasmidpGETrec which expresses the enzymes necessary for E/T cloning afterarabinose induction (Schumacher et al., 2000). Kanamycin-resistantcolonies were picked, DNA was isolated and subjected to Southern blotanalysis using a kan^(R)-specific probe. In both gD-negative RacH-BAC(c) and gM-negative RacH-BAC (d), fragments of the expected sizes (gD:20.4 kbp; gM: 9.3 kbp specifically reacted with the kan^(R) probe.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Before the embodiments of the present invention it must be notedthat as used herein and in the appended claims, the singular forms “a”,“an”, and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a virus” includes aplurality of such viruses, reference to the “cell” is a reference to oneor more cells and equivalents thereof known to those skilled in the art,and so forth. Unless defined otherwise, all technical and scientificterms used herein have the same meanings as commonly understood by oneof ordinary skill in the art to which this invention belongs. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods, devices, and materials are now described. Allpublications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the cell lines, vectors, andmethodologies which are reported in the publications which might be usedin connection with the invention. Nothing herein is to be construed asan admission that the invention is not entitled to antedate suchdisclosure by virtue of prior invention.

[0018] The invention relates to an artificial chromosome vectorcharacterized in that it comprises essentially the entire genome of anEHV strain from which infectious progeny can be reconstituted aftertransfection into a permissive cell.

[0019] With the artificial chromosome vectors according to the presentinvention, safe EHV-vaccines comprising EHV with defined attenuationscan be generated. Such viruses are useful for the preparation of a safelive vaccine for use in the prevention and/or treatment of EHVinfections (see infra). The invention provides the possibility for afast and efficient manipulation of the EHV genome which remains fullyinfectious for eukaryotic cells or is modified into areplication-deficient virus. There was a long lasting need in the artfor such a tool to handle and manipulate the huge genome of EHV. Lastly,the EHV nucleic acid can be used as a polynucleotide vaccine which isapplied either topically or systemically to naive or primed horses andmay also be applied in utero.

[0020] The present invention is illustrated in example 1 showing thecloning of the entire genome of EHV-1 as an infectious mini F plasmid(‘bacterial artificial chromosome’, BAC) into Escherichia coli. Thegeneration of said BAC was not trivial and was posed many difficulties,including the preparation and extraction of sufficient amounts ofcircular DNA. The circularized form of recombinant viral DNA was neededto transform DH10B cells with the recombinant DNA in order to preparethe mini F plasmid-cloned EHV DNA. To obtain sufficient amounts ofcircular viral DNA, early viral transcription was blocked by theaddition of 100 μg per ml of cycloheximide after infection of cells.Viral DNA was then prepared and used for transformation of DH10B cells.Only from cells treated with cycloheximide was it possible to extractsufficient amounts of circular DNA and to obtain DH10B clones containingthe enitre RacH genome.

[0021] “Essentially” means that the EHV genome is complete with theexception that it may carry a mutation as set out infra.

[0022] “Artificial chromosome” relates to any known artificialchromosomes, such as yeast, or preferably bacterial artificialchromosomes.

[0023] Preferably, a bacterial artificial chromosome (BAC) according tothe invention is a vector used to clone large DNA fragments (100- to300-kb insert size) in Escherichia coli cells which is based onnaturally occurring F-factor plasmid found in the bacterium E. coli(Shizuya, H., B. Birren, U. J. Kim et al. 1992. Cloning and stablemaintenance of 300-kilobase-pair fragments of human DNA in Escherichiacoli using an F-factor-based vector. Proceedings National Academy ofScience 89: 8794-8797). The type of vector is preferably based on aF-plasmid replicon containing the origin of replication (oriS) and itsown DNA polymerase (repE) as well as the genes parA and parB involved inmaintaining its copy number at a level of one or two per E. coli. Theantibiotic resistance marker is preferably Cm-resistance.

[0024] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV is EHV-1.

[0025] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV isEHV-4.

[0026] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis RacH.

[0027] According the invention, any type of mutation can be introducedinto the EHV genome, in order to obtain a replication-deficient and/orattenuated EHV virus. Such mutations include, but are not limited to anymutation (e.g. deletion, insertion, substitution) relating to theglycoproteins gB, gC, gD, gE, gG, gI, gJ, gL and gM, gp1/2 and anycombination thereof. Preferably, said mutations are deletion mutations,i.e. the respective glycoproteins such as e.g. gM are completelydeleted.

[0028] Thus, the invention preferably relates to an artificialchromosome vector according to the invention, characterized in that theEHV strain is lacking the glycoprotein gB.

[0029] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gC.

[0030] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gD.

[0031] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gE.

[0032] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gG.

[0033] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gH.

[0034] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gI.

[0035] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gK.

[0036] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gL.

[0037] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gM.

[0038] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gp1/2.

[0039] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the artificialchromosome is a bacterial artificial chromosome (BAC). Said BAC's can bepropagated in any bacterium known to the skilled person, e.g andpreferably Escherichia coli.

[0040] The invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the artificialchromosome is a yeast artificial chromosome (YAC).

[0041] The invention preferably relates to an artificial chromosomevector RacH-BAC according to the invention, characterized in that theartificial chromosome as deposited under the accession number ECACC01032704 with the ECACC in Porton Down, UK (European Collection of CellCultures, CAMR, Salisbury, Wiltshire SP4 0JG, UK).

[0042] Another important embodiment of the present invention is apolynucleotide vaccine encoding an an artificial chromosome vector orEHV contained therein according to the invention.

[0043] Yet another important embodiment of the present invention is theuse of an artificial chromosome vector according to the invention forthe generation of infectious EHV.

[0044] The invention furthermore relates to a method for the generationof an infectious EHV, characterized in that an artificial chromosomevector according to the invention is used to infect a suitable cell lineand the shedded virus is collected and purified.

[0045] The invention furthermore relates to a method for the generationof an attenuated EHV, characterized in that the EHV sequence containedin an artificial chromosome vector according to the invention isspecifically modified by molecular biology techniques.

[0046] Said modifications may be carried out by methods known in theart, e.g. site directed mutagenesis see e.g. Sambrook et al.(1989)Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

[0047] Furthermore, the invention relates to a EHV obtainable by amethod according to the invention.

[0048] Another very important embodiment is a pharmaceutical compositioncomprising a polynucleotide according to the invention and optionallypharmaceutically acceptable carriers and/or excipients. Such apolynucleotide according to the invention may also be used in apharmaceutical composition within the scope of this invention, e.g. forDNA vaccination.

[0049] One example of a targeted system of administration, e.g. forpolynucleotides according to the invention is a colloidal dispersionsystem. Colloidal dispersion systems comprise macromolecule complexes,nanocapsules, microspheres and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles and liposomes orliposome formulations. Liposomes are the preferred colloidal systemaccording to the invention. Liposomes are artificial membrane vesicleswhich are useful as carriers in vitro and in vivo. These formulationsmay carry a cationic, anionic or neutral charge. It has been shown thatlarge unilamellar vesicles (LUV) ranging from 0.2-4.0 μm in size mayenclose a major part of an aqueous buffer solution with largemacromolecules. RNA, DNA and intact virions can be encapsulated in theaqueous phase inside and transported to the target in a biologicallyactive form (Fraley R et al., 1981, Trends Biochem Sci 6, 77-80). Inaddition to mammalian cells, liposomes have also proved suitable for thetargeted transporting of nucleotides into plant, yeast and bacterialcells. In order to be an efficient gene transfer carrier the followingproperties should be present: (1) the genes should be enclosed with highefficiency without reducing their biological activity; (2) there shouldbe preferential and substantial binding to the target cell compared withnon-target cells; (3) the aqueous phase of the vehicle should betransferred highly efficiently into the target cell cytoplasm; and (4)the genetic information should be expressed accurately and efficiently(Mannino R J et al., 1988, BioTechniques 6, 682-690).

[0050] The composition of the liposomes usually consists of acombination of phospholipids, particularly high phase transitiontemperature phospholipids, e.g. combined with steroids such ascholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of the liposomes depend on the pH, the ionconcentration and the presence of divalent cations.

[0051] The pharmaceutical composition according to the invention mayalso contain a vector according to the invention, e.g. a BAC vectorcomprising an EHV genome as described supra, as a naked “gene expressionvector”. This means that the vector according to the invention is notassociated with an adjuvant for targeted administration (e.g. liposomes,colloidal particles, etc.). A major advantage of naked DNA vectors isthe absence of any immune response caused by the vector itself

[0052] The EHV nucleic acid can be used as a polynucleotide vaccine (seepharmaceutical composition, supra) which is applied either topically(e.g. intranasally) or systemically to naive or primed horses and mayalso be applied in utero.

[0053] Another very important embodiment is a pharmaceutical compositioncomprising an EHV virus according to the invention and pharmaceuticallyacceptable carriers and/or excipients. A pharmaceutically acceptablecarrier can contain physiologically acceptable compounds that act, forexample, to stabilize or to increase the absorption or form part of aslow release formulation of the EHV virus or the polynucleotideaccording to the invention. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers or excipients(see also e.g. Remington's Pharmaceutical Sciences (1990). 18th ed. MackPubl., Easton). One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition.

[0054] Furthermore, the invention relates to the use of a polynucleotideaccording to the invention in the manufacture of a vaccine for theprevention and/or treatment of EHV infections.

[0055] Furthermore, the invention relates to the use of an EHV virusaccording to the invention in the manufacture of a vaccine for theprevention and/or treatment of EHV infections.

[0056] Furthermore, the invention relates to the use of the BACtechnology to establish a highly virulent and genetically wellcharacterized EHV which can be used for immunization and challengestudies for use e.g. in vaccine potency studies.

[0057] Furthermore, the invention relates to the use of EHV BACsaccording to the invention to generate mutant BACs that are generatedtaking into account appearing genetic or antigenetic variants of EHV.This relates to one or more mutations present withing, new variants' ofEHV which can be easily introduced in the existing EHV BAC.

[0058] The following example is intended to aid the understanding of theinvention and should in no way be regarded as limiting the scope of theinvention.

EXAMPLE 1 Construction of an EHV-1 Bacterial Artificial Chromosome

[0059] A genetically uniform population of RacH (256^(th) passage) wasisolated. With RacH, passage 257, Rk13 cells were infected and a motherpool was established. Virus of one additional passage on RK13 cells wasused to infect RK13 cells, from which viral DNA was prepared. Tenmicrograms (μg) of viral DNA were co-transfected with 10 μg of plasmidp71-pHA2 (FIG. 1) into RK13 cells. For construction of plasmid p71-pHA2,2.0 and 2.4 kbp fragments on either side of the EHV-1 gene 71 (FIG. 1;Table 1) were amplified by polymerase chain reaction (PCR) using primerscontaining appropriate restriction enzyme sites (Table 1). Bothfragments were subsequently cloned into pTZ18R (Pharmacia-Amersham) toobtain plasmid p71 (FIG. 1). A BAC vector (pHA2; Messerle et al., 1997)containing the Eco-gpt and GFP (green flourescent protein) genes underthe control of the HCMV (human cytomegalovirus) immediate early promoterwas released as a PacI fragment from plasmid pHA2 and inserted into thePacI sites of the 2.0 and 2.4 kbp fragment cloned in p71 (FIG. 1; Table1). Virus progeny was harvested and individual plaques expressing thegreen fluorescent protein (GFP) were isolated and subjected to threerounds of plaque purification until virus progeny stained homogenouslygreen under the fluorescent microscope (Seyboldt et al., 2000).Similarly, co-transfections of p71-pHA2 and DNA of EHV-1 strain KentuckyA (KyA) were performed and the recombinant virus was purified tohomogeneity. Recombinant virus DNA was prepared (Schumacher et al.,2000) and electroporated into Escherichia coli strain DH10B (Messerle etal., 1997; Schumacher et al., 2000). Electrocompetent bacteria wereprepared as described (Muyrers et al., 1999; Narayanan et al., 1999;Zhang et al., 1998) and electroporation was performed in 0.1 cm cuvettesat 1250 V, a resistance of 200 Ω, and a capacitance of 25 μF (Easyjectelectroporation system, Eurogenentec). Transformed bacteria wereincubated in 1 ml of Luria-Bertani (LB) medium (28) supplemented with0.4% glucose for 1 hr at 37° C., and then plated on LB agar containing30 μg/ml chloramphenicol. Single colonies were picked into liquid LBmedium, and small scale preparations of BAC DNA were performed byalkaline lysis of Escherichia coli (Schumacher et al., 2000). Largescale preparation of BAC DNA was achieved by silica-based affinitychromatography using commercially available kits (Qiagen, Macherey &Nagel).

[0060] From the chloramphenicol-resistant bacterial colonies, one colonyeach was chosen and named RacH-BAC which contained the EHV-1 RacHgenome. RACH-BAC DNA was cleaved with restriction enzymes BamHI, EcoRIand HindIII and the restriction enzyme patterns were compared to thoseof parental viral DNA. (Schumacher et al., 2000). The calculated andexpected changes in the banding pattern after insertion of the mini Fplasmid into the gene 71 locus were observed in RacH-BAC. In contrast,no other differences in restriction enzyme patterns as compared to theparental virus were obvious (FIG. 2). After purification of RacH-BAC DNAusing affinity chromatography, RK13 cells were transfected with 1 μg ofrecombinant DNA. At one day after transfection, foci of greenfluorescent cells were visible which developed into plaques on thefollowing days after infection (FIG. 3). From these results we concludedthat the RacH strain of EHV-1 was cloned as an infectious full-lengthviral DNA in Escherichia coli. Deletion of gene 71 in RacH-BAC resultedin a less than 10% reduction in plaque size (FIG. 3). TABLE 1 Fragmentor plasmid Primer Sequence generated Gen71 1.Fr.5′-GCAggtaccTTTGCACAACTTTAGGATGAC-3′ 2.0-kb flank for p71-pHA2 for Gen711.Fr. 5′-GATggatccCTttaattaaGTAGACGCGGCTGTAGTAAC-3′ 2.0-kb flank forp71-pHA2 rev Gen71 2.Fr. 5′-ACAgtcgacCTttaattaaTCGGGGAACTAGTCACACTC-3′2.4-kb flank for p71-pHA2 for Gen71 2.Fr.5′-CGAgcatgcAGTTTTACGCGAAGGATATAC3′ 2.4-kb flank for p71-pHA2 rev Kan950for 5′-GCCAGTGTTACAACCAATTAACC-3′ Kan^(r)950 gene Kan950 rev5′-CGATTTATTCAACAAAGCCACG-3′ Kan^(r)950 gene gM950EHV5′-GGTTTCAAATTCCTCGCTCACCACGTCGTAAATTGGCTCT Kan^(r)950 gene for gM forTCTGCGTCCG GCCAGTGTTACACCAATTAAAC-3′ deletion gM950EHV5′-AAAACCACAGCGTGGTCGATGGAGTGTGGATGCGGCAG Kan^(r)950 gene for gM revATAGCTGGTGGA CGATTTATTCAACAAAGCCACG-3′ deletion gD-950 for5′-CGCCCACTCAACTTCCAACTTCGCTTTAGTGGCTGCGACC Kan^(r)950 gene for gDACGCTAACAG CGATTTATTCAACAAAGCCACG-3′ deletion gD-950-1 rev5′-TTCTTCCGACGCAAGCAGACGTATAGAATGACGCCCACC Kan^(r)950 gene for gDAATACTAG GCCAGTGTTACAACAAATTAACC-3′ deletion

Mutagenesis of EHV-1 BACs

[0061] For mutagenesis of RacH-BAC DNA in Escherichia coli, recE- andrecT-catalyzed reactions promoting homologous recombination betweenlinear DNA fragments, also referred to as E/T cloning, was performed(Muyrers et al., 1999; Zhang et al., 1999). Plasmid pGETrec (kindlyprovided by Dr. Panos Ioannou, Murdoch Institute, Melbourne, Australia)harboring the recE, recT and bacteriophage λ gam gene (Narayanan et al.,1999) was transformed into RacH-BAC-containing DH10B cells. Afterinduction of recE, recT and gam by addition of 0.2% arabinose,electrocompetent cells were prepared essentially as described (Muyrerset al., 1999). To delete the gD and gM gene in RACH-BAC, the kanamycinresistance gene (kan^(R)) of plasmid pACYC177 (Stratagene) was amplifiedby PCR. The designed primers contained 50 nucleotide homology armsbordering the desired deletion within gD or gM and 20 nucleotides foramplification of kan^(R) (Table 1). The resulting 0.95 kbp fragment waspurified from an agarose gel (Qiagen) and electroporated intopGETrec-containing RacH-BAC cells. Colonies harboring the cam^(R) andkan^(R) genes were identified on plates containing both antibiotics.

[0062] H-BACΔgD and H-BACΔgM DNA were isolated from Escherichia coli bychromatography and subjected to restriction enzyme digestion andSouthern blot analysis (FIG. 4) transfection studies were performed.Whereas RacH-BAC and H-BACΔgM were able to induce viral plaques on RK13cells, H-BACΔgD was able to induce plaques on cells expressing gD intrans only. The gD cells transiently expressed EHV-1 gD aftertransfection of a recombinant plasmid in which gD is under control ofthe HCMV immediate early promoter/enhancer. These observations indicatedthat EHV-1 gD is essential for virus growth in vitro.

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What is claimed is:
 1. Bacterial artificial chromosome vectorcharacterized in that it comprises essentially the entire genome of anEHV strain.
 2. The artificial chromosome vector according to claim 1,characterized in that the EHV is EHV-1.
 3. The artificial chromosomevector according to claim 1, characterized in that the EHV is EHV-4. 4.The artificial chromosome vector according to claim 1, characterized inthat the EHV strain is RacH.
 5. The artificial chromosome vectoraccording to claim 4, chraterized in that it is the vector with theaccession No. ECACC
 01032704. 6. The artificial chromosome vectoraccording to claim 1, characterized in that the EHV strain is lackingthe glycoprotein gB.
 7. The artificial chromosome vector according toclaim 1, characterized in that the EHV strain is lacking theglycoprotein gC.
 8. The artificial chromosome vector according to claim1, characterized in that the EHV strain is lacking the glycoprotein gD.9. The artificial chromosome vector according to claim 1, characterizedin that the EHV strain is lacking the glycoprotein gE.
 10. Theartificial chromosome vector according to claim 1, characterized in thatthe EHV strain is lacking the glycoprotein gG.
 11. The artificialchromosome vector according to claim 1, characterized in that the EHVstrain is lacking the glycoprotein gH.
 12. The artificial chromosomevector according to claim 1, characterized in that the EHV strain islacking the glycoprotein gI.
 13. The artificial chromosome vectoraccording to claim 1, characterized in that the EHV strain is lackingthe glycoprotein gK.
 14. The artificial chromosome vector according toclaim 1, characterized in that the EHV strain is lacking theglycoprotein gL.
 15. The artificial chromosome vector according to claim1, characterized in that the EHV strain is lacking the glycoprotein gM.16. The artificial chromosome vector according to claim 1, characterizedin that the EHV strain is lacking the glycoprotein gp1/2.
 17. Apolynucleotide encoding an artificial chromosome vector, which vector ischaracterized in that it comprises essentially the entire genome of anEHV strain, or EHV contained in the vector.
 18. A method for generatinginfectious EHV which comprises using an artificial chromosome vector,which vector is characterized in that it comprises essentially theentire genome of an EHV strain.
 19. A method for generating infectiousEHV which comprises using the polynucleotide as according to claim 18.20. A method for generating EHV which comprises infecting a suitablecell line with the artificial chromosome vector according to claim 1,allowing the vector to replicate and shed virus, collecting the shedvirus and purifying the collected virus.
 21. A method for generating anattenuated EHV which comprises modifying by molecular biology techniquesthe EHV sequence contained in an artificial chromosome vector accordingto claim
 1. 22. The method according to claim 22 wherein a foreignsequence of another viral, bacterial or parasitic pathogen is added tothe artificial chromosome vector.
 23. A method for generating a virulentEHV which comprises modifying by molecular biology techniques the EHVsequence contained in an artificial chromosome vector according toclaim
 1. 24. The method according to claim 23 wherein a foreign sequenceof another viral, bacterial or parasitic pathogen is added to theartificial chromosome vector.